A self-designed CpG ODN enhanced the anti-melanoma effect of pimozide
Huijie JiaJing GuoPingping WangKe SunJian ChenWenjing RenWei TianYunfan YangJie LiXiaoming LiuRuipeng LiJiateng ZhongMingyong WangZhongwei TianZhiwei FengTiesuo Zhao
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Keywords:
Pimozide
CpG Oligodeoxynucleotide
TLR9
TLR9
Toll-Like Receptor 9
CpG Oligodeoxynucleotide
CpG site
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To determine the role of Toll-like receptor 9 (TLR9) in synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides (CpG-ODN)-induced species-specific immune responses.CpG-ODN was co-cultured with peripheral blood mononuclear cells (PBMC) of humans, macaques and mice. The IFN-alpha in the supernatant was measured by ELISA. The reverse transcription PCR was used to analyze the expression levels of TLR9 mRNA in PBMC.CpG-ODN induced high amounts of IFN-alpha in human PBMC, but had no effect on macaques and mice. The expression of TLR9 mRNA was observed in all human PBMC, and the levels of TLR9 mRNA were significantly up-regulated with the stimulation of CpG-ODN. We did not observe any expression of TLR9 mRNA in PBMC in macaques.TLR9 underlies the molecular foundation of CpG-ODN-induced species specificity.
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Toll-Like Receptor 9
CpG site
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Abstract The Toll-like receptor (TLR)9 is critical for the recognition of immunostimulatory CpG motifs but may cooperate with other TLRs. We analyzed TLR1–10 mRNA expression by using quantitative real-time PCR in highly purified subsets of human PBMC and determined the sensitivity of these subsets to CpG oligodeoxynucleotides (ODN). TLR1 and TLR6 were expressed in all cell types examined. TLR10 was highly expressed in B cells and weakly expressed in plasmacytoid dendritic cells (PDC). High expression of TLR2 was characteristic for monocytes. PDC and B cells expressed marked levels of TLR7 and TLR9 and were directly sensitive to CpG ODN. In CpG ODN-stimulated PDC and B cells, TLR9 expression rapidly decreased, as opposed to TLR7, which was up-regulated in PDC and decreased in B cells. In monocytes, NK cells, and T cells, TLR7 was absent. Despite low expression of TLR9, monocytes, NK cells, and T cells did not respond to CpG ODN in the absence of PDC but were activated in the presence of PDC. In conclusion, our studies provide evidence that PDC and B cells, but not monocytes, NK cells, or T cells, are primary targets of CpG ODN in peripheral blood. The characteristic expression pattern of TLR1–10 in cellular subsets of human PBMC is consistent with the concept that TLR9 is essential in the recognition of CpG ODN in PDC and B cells. In addition, selective regulation of TLR7 expression in PDC and B cells by CpG ODN revealed TLR7 as a candidate TLR potentially involved in modulating the recognition of CpG motifs.
TLR9
CpG Oligodeoxynucleotide
TLR7
CpG site
Plasmacytoid dendritic cell
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Toll-like receptor (TLR) 9 is an endosomal receptor recognizing bacterial DNA/CpG-containing oligodeoxynucleotides (CpG ODN). Blocking CpG ODN/TLR9 activity represents a strategy for therapeutic prevention of immune system overactivation. Herein, we report that a synthetic peptide (SP) representing the leucine-rich repeat 11 subdomain of the human TLR9 extracellular domain could attenuate CpG ODN/TLR9 activity in RAW264.7 cells by binding to CpG ODN and decreasing its internalization. Our results demonstrate that preincubation with SP specifically inhibited CpG ODN- but not lipopolysaccharide (LPS)- and lipopeptide (PAM3CSK4)-stimulated TNF-α and IL-6 release. Preincubation of SP with CpG ODN dose-dependently decreased TLR9-driven phosphorylation of IκBα and ERK and activation of NF-κB/p65. Moreover, SP dose-dependently decreased FAM-labeled CpG ODN internalization, whereas non-labeled CpG ODN reversed the inhibition. The KD value of SP-CpG ODN binding was within the micromolar range. Our results demonstrated that SP was a specific inhibitor of CpG ODN/TLR9 activity via binding to CpG ODN, leading to reduced ODN internalization and decreased activation of subsequent pathways within cells. Thus, SP could be used as a potential CpG ODN antagonist to block TLR9 signaling.
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CpG Oligodeoxynucleotide
Internalization
Toll-Like Receptor 9
CpG site
Lipopeptide
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TLR9のリガンドであるCpG ODNは、免疫アジュバントとしてがんや感染症に対する治療への利用が期待されている。筆者らは、ナノ構造化核酸を基盤とする研究を展開し、CpG ODNの立体構造と細胞との相互作用、アジュバント活性との相関について検討してきた。その結果、TLR9発現細胞によるナノ構造化核酸の取り込みにおけるMSR1の関与を見出した。また、さまざまな構造的特徴のナノ構造化核酸を作製し、これがCpG ODNによるTLR9発現細胞からのサイトカイン産生増大に有用であることを示した。さらには、ナノ構造化核酸を連結することで得られるDNAハイドロゲルが、自然免疫活性化作用を有しCpG ODNを徐放するデリバリーシステムとしてがん治療に有効であることを明らかにした。
TLR9
CpG Oligodeoxynucleotide
CpG site
Toll-Like Receptor 9
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TLR9
CpG Oligodeoxynucleotide
Toll-Like Receptor 9
CpG site
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TLR9
CpG Oligodeoxynucleotide
CpG site
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CpG-oligodeoxynucleotides (CpG-ODN) are potent immune stimuli being developed for use as adjuvants in different species. Toll-like receptor 9 (TLR9) is the cellular receptor for CpG-ODN in mammalian cells. The CpG-ODN with 18–24 deoxynucleotides that are in current use for human and mouse cells, however, have low activity with rabbit TLR9. Using a cell-based activation assay, we developed a type of CpG-ODN containing a GACGTT or AACGTT motif in 12 phosphorothioate-modified deoxynucleotides with potent stimulatory activity for rabbit TLR9. The developed CpG-ODN have higher activities than other developed CpG-ODN in eliciting antigen-nonspecific immune responses in rabbit splenocytes. When mixed with an NJ85 peptide derived from rabbit hemorrhagic disease virus, they had potent activities to boost an antigen-specific T cell activation and antibody production in rabbits. Compared to Freund's adjuvant, the developed CpG-ODN are capable of boosting a potent and less toxic antibody response. The results of this study suggest that both the choice of CpG-motif and its length are important factors for CpG-ODN to effectively activate rabbit TLR9 mediated immune responses.
TLR9
CpG Oligodeoxynucleotide
Toll-Like Receptor 9
CpG site
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CpG ODN, a synthetic oligodeoxynucleotide containing CpG motifs, is a TLR9 agonist as it could be recognized by Toll-like receptor 9 (TLR9) expressed on the membranes of B cells, dendritic cells, monocytes and macrophages. CpG ODN is the kind of vaccine adjuvant that promotes Th1 immune responses and enhances the immunogenicity of vaccines with high safety, showing promising prospects in the prevention and treatment of infections, tumors and allergic diseases. This review mainly focuses on the introduction, classification and structure features, immune mechanism, safety and clinical application of CpG ODN as vaccine adjuvant in order to provide references for further researches and application of CpG ODN as immune adjuvant.
Key words:
Immune adjuvant; CpG ODN; Vaccine; Immune response
TLR9
CpG Oligodeoxynucleotide
CpG site
Toll-Like Receptor 9
Vaccine adjuvant
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Unmethylated cytidine-phosphate-guanosine-containing oligodeoxynucleotides(CpG ODN) are Toll-like receptor 9(TLR9) agonists.TLR9 was identified as the mediator of the immunstimulatory effects of CpG ODN.Structure-activity relationship studies of CpG ODN has defined 4 categories: K,D,C and P types of CpG ODN with distinct structural and biological characteristics.They were potent activators of plasmacytoid dentritic cells(pDCs) and B cells,respectively.Over the last decade,experiments and clinical trials have been conducted to treat non-small-cell lung cancer with CpG ODN alone or combined with chemotherapy,radiotherapy,targeting agents and vaccines.Immunoenhancement was observed in these tests.The safety of CpG ODN was also described briefly.
CpG Oligodeoxynucleotide
TLR9
Cytidine
CpG site
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